Antisense oligonucleotide against human acetylcholinesterase (AChE) and uses thereof

ABSTRACT

The invention relates to an antisense oligonucleotide targeted to the coding region of the human acetylcholinesterase (AChE), which selectively suppresses the AChE-R isoform of the enzyme. The antisense oligonucleotide is intended for use in the treatment and/or prevention of neuromuscular disorders, preferably myasthenia gravis. In addition, it can penetrate the blood-brain barrier (BBB) and destroy AChE-R within central nervous system neurons, while also serving as a carrier to transport molecules across the BBB.

FIELD OF THE INVENTION

The present invention relates to a synthetic antisenseoligodeoxynucleotide targeted to the common coding domain of humanacetylcholinesterase (AChE) mRNA, and to pharmaceutical or medicalcompositions comprising the same, particularly for the treatment and/orprevention of a progressive neuromuscular disorder.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Neuromuscular junctions (NMJ) are highly specialized, morphologicallydistinct, and well-characterized cholinergic synapses [Hall and Sanes(1993) Cell 72 Suppl., 99-121]. Chronic impairments in NMJ activityinduce neuromuscular disorders characterized by progressivedeterioration of muscle structure and function. The molecular andcellular mechanisms leading from compromised NMJ activity to musclewasting have not been elucidated.

One such disorder is myasthenia gravis (MG), caused by a defect inneuromuscular transmission mediated by auto-antibodies that severelyreduce the number of functional post-synaptic muscle nicotinicacetylcholine receptors (nAChR) [Drachman D. G. (1994) N. Engl. J. Med.330, 1797-1810; Vincent A. (1999) Curr. Opin. Neurol. 12, 545-551]. MGis characterized by fluctuating muscle weakness that may be transientlyimproved by inhibitors of acetylcholinesterase (AChE) [Penn A. S. andRowland L. P. (1995) Myasthenia Gravis In: Meritt's Textbook ofNeurology, 9^(th) Edition, Williams and Wilkins, Baltimore, sectionXVII, 754-761]. The characteristic electrodiagnostic abnormality is aprogressive, rapid, decline in the amplitude of compound muscle actionpotentials (CMAP) evoked by repetitive nerve stimulation at 3 or 5 Hz.To date, the standard treatment for MG includes immunosuppressivetherapy combined with chronic administration of multiple daily doses ofperipheral AChE inhibitors such as pyridostigmine (Mestinon™). WhileAChE inhibitors effectively restore muscle performance in MG patients,their effects are short-lived, calling for the development of additionaleffective treatment.

Antisense technology offers an attractive, gene-based alternative toconventional anti-cholinesterase therapeutics. Antisense technologyexploits the rules of Watson-Crick base pairing to design shortoligonucleotides, 15-25 residues in length, whose sequence iscomplementary to that of a target mRNA [Agrawal S, and Kandimalla E. R.(2000) Mol. Med. Today, 6, 72-81]. Stretches of double-stranded RNA,resulting from hybridization of the antisense oligonucleotide (ASON)with its target, activate RNAse H [Crooke S. T. (2000) Methods Enzymol.313, 3-45] and promote specific degradation of the duplex mRNA. Asantisense therapeutics target RNA rather than proteins, they offer thepotential to design highly specific drugs with effective concentrationsin the nanomolar range [Galyam N. et al. (2001) Antisense Nucleic AcidDrug Dev. 11, 51-57]. Phosphorothioated and 3′ terminally protected2′-O-methyl antisense oligonucleotides targeted to mouse AChE mRNA wereshown to be effective in blocking AChE expression in vitro in culturedhuman and rodent cells [Koenigsberger C. et al. (1997) J. Neurochem. 69,1389-1397; WO 98/26062; Grisaru D. et al. (2001) Mol. Med. 7, 93-105],and in vivo in brain [Shohami E. et al. (2000) J. Mol. Med. 78, 278-236;Cohen et al. (2002) Molecular Psychiatry, in press], muscle [Lev-LehmanE. et al. (2000) J. Mol. Neurosci. 14, 93-105] and bone marrow [Grisaruet al. (2001) ibid.].

The inventors have recently observed that treatment with theirreversible cholinesterase inhibitor diisopropylfluorophosphonate (DFP)induces overexpression of an otherwise rare, non-synaptic alternativesplicing variant of AChE, AChE-R, in brain [Kaufer D. et al. (1998)Nature, 393, 373-377] and intestine [Shapira M. et al. (2000) Hum. Mol.Genet. 9, 1273-1282]. Muscles from animals treated with DFP alsooverexpressed AChE-R, accompanied by exaggerated neurite branching,disorganized wasting fibers and proliferation of NMJs. Partiallyprotected 2′-O-methyl antisense oligonucleotides targeted to mouse AChEmRNA suppressed feedback upregulation of AChE and amelioratedDFP-induced NMJ proliferation [Lev-Lehman et al. (2000) ibid.]. Theseobservations demonstrated that cholinergic stress elicits overexpressionof AChE-R in muscle and that antisense oligonucleotides can suppresssuch AChE-R excess and prevent its deleterious outcome.

As mentioned above, the characteristic electrodiagnostic abnormality isa progressive, rapid decline in the amplitude of muscle actionpotentials evoked by repetitive nerve stimulation at 3 or 5 Hz. Thismyasthenic fatigue is caused by decrease in the number of AChR moleculesavailable at the post-synaptic site. Inhibiting anti-AChR antibodies arepresent in 85% to 90% of patients [Vincent, A. (1999) id ibid].

Patients with MG, but not with congenital myasthenias due to othercauses [Triggs et al. (1992) Muscle Nerve 15, 267-72], display atransient clinical response to AChE inhibitors such as edrophonium. Theavailable anti-AChE drugs are the first line of treatment, but mostpatients require further help. This includes drastic measures, such asplasma exchange, thymectomy and immunosuppression. Unfortunately, all ofthe currently employed MG drug regimens. are associated with deleteriouslong-term consequences. These include disturbance of neuromusculartransmission, exacerbation and induction of MG symptoms. Also, theotherwise safe use of common drugs such as anti-infectives,cardiovascular drugs, anticholinergics, anticonvulsants, antirheumaticsand others has been reported to worsen the symptoms of MG patients[Wittbrodt (1997) Arch. Intern. Med., 157, 399-408].

While the neuromuscular malfunctioning associated with MG can betransiently alleviated by systemic chronic administration of carbamateacetylcholinesterase (AChE) inhibitors (e.g. pyridostigmine), theinventors have found that pyridostigmine induces a feedback responseleading to excess AChE accumulation [Friedman et al. (1996) NatureMedicine 2, 1382-1385; Kaufer et al. (1998) id ibid; Meshorer, E. et al.(2002) Science 295, 508-12]. This suggested that the chronic use of suchinhibitors would modify the cholinergic balance in the patients'neuromuscular system and would require increased doses of these drugs;it also provided an explanation of the highly variable dose regimenemployed in MG patients; and it called for the development of analternative approach to suppress acetylcholine hydrolysis.

AChE-encoding RNA is subject to 3′ alternative splicing yielding mRNAsencoding a “synaptic” (S) isoform, containing exons 1-4 and 6, alsodesignated E6 mRNA herein, an “erythrocytic” (E) isoform, containingexons 1-6, also designated E5 mRNA herein, and the “readthrough” AChE-Rderived from the 3′-unspliced transcript, containing exons 1-6 and thepseudo-intron I4, also designated I4 mRNA herein.

Transgenic mice overexpressing human AChE-S in spinal cord motoneurons,but not in muscle, displayed progressive neuromotor impairments thatwere associated with changes in NMJ ultrastructure [Andres, C. et al.(1997) Proc. Natl. Acad. Sci. USA 94, 8173-8178]. However, it was notclear whether the moderate extent of overexpressed AChE in muscle wasitself sufficient to mediate this severe myopathology. In rodent brain,the inventors found previously that both traumatic stress andcholinesterase inhibitors induce dramatic calcium-dependentoverexpression of AChE-R [Kaufer, et al. (1998) id ibid.], associatedwith neuronal hypersensitivity to both cholinergic agonists andantagonists [Meshorer et al. (2002) id ibid].

Chronic AChE excess was found to cause progressive neuromotordeterioration in transgenic mice and amphibian embryos [Ben Aziz-Aloyaet al. (1993) Proc. Natl. Acad. Sci. USA, 90, 2471-2475; Seidman et al.(1994) J. Neurochem. 62, 1670-1681; Seidman, et al. (1995) Mol. Cell.Biol. 15, 2993-3002; Andres, C. et al. (1997) Proc. Natl. Acad. Sci. USA94, 8173-8178; Sternfeld et al. (1998) J. Neurosci. 18, 1240-1249].Also, myasthenic patients suffer acute crisis events, with a reportedaverage annual incidence of 2.5% [Berrouschot et al. (1997) Crit. CareMed. 25, 1228-35] associated with respiratory failure reminiscent ofanti-AChE intoxications.

In one approach, the prior art teaches that chemically protected RNAaptamers capable of blocking the autoantibodies to the nicotinicAcetylcholine Receptor (nAChR) may be developed and used to treat MG.This approach has several drawbacks in that the RNA aptamers do not havethe amplification power characteristic of the RNAse-inducing antisenseagents and in that it fails to address the problem of the feedbackresponses in MG.

The present inventors have previously found that antisenseoligonucleotides against the common coding region of AChE are useful forsuppressing AChE production [WO 98/26062]. This publication also teachesthat antisense oligonucleotides against the human AChE are useful in thetreatment of memory deficiencies as observed in transgenic mice thatexpressed human AChE in their brain. The observed effects (see Table 4-5in WO 98/26062) are similar in their effect, yet considerably longer inthe duration of their action than the prior art AChE inhibitor tacrine(see FIG. 9B in WO 98/26062).

In view of the above, it is desirable to further improve the treatmentapproaches for MG and other diseases involving impairment inneuromuscular transmission. The prior art treatment involving the use ofAChE inhibitors is afflicted with undesirable side effects because ofthe induction of AChE and neuromuscular impairments by such inhibitors;and because it is subject to variable efficacy under altered mentalstate (stress).

WO01/36627 teaches that morphological and functional changes in the NMJcorrelate with overexpression of a specific isoform of AChE mRNA, viz.,the “readthrough” isoform containing the pseudo-intron 14 in the maturemRNA. Said PCT application also shows that antisense oligonucleotidesdirected to the common coding region of AChE may be used to specificallydestroy AChE-R mRNA, and that AChE antisense agents are by far superiorto conventional AChE enzyme inhibitor drugs in the treatment ofneuromuscular disorders. The superiority of these antisense agents maybe due to the fact that conventional enzyme inhibitors actively induceI4 AChE mRNA overexpression. According to the teachings of WO01/36627,this may lead to detrimental changes in the NMJ. This consequence oftreatment may be entirely avoided by using the antisense agents ofWO01/36627.

The Blood-Brain Barrier (BBB) maintains a homeostatic environment in thecentral nervous system (CNS). The capillaries that supply the blood tothe brain have tight junctions which block the passage of most moleculesthrough the capillary endothelial membranes. While the membranes doallow passage of lipid soluble materials, water soluble materials do notgenerally pass through the BBB. Mediated transport mechanisms exist totransport the water soluble glucose and essential amino acids throughthe BBB. Active support mechanisms remove molecules which become inexcess, such as potassium, from the brain [for general review see Betzet al., Blood-Brain-Cerebrospinal Fluid Barriers, Chapter 32, in BasicNeurochemistry, 5^(th) ed., Eds Siegel, Albers Agranoff, Molinoff, pp.681-701; Goldstein and Betz (1986) Scientific American, September, pp.74-83].

The BBB impedes the delivery of drugs to the CNS. Methods have beendesigned to deliver needed drugs such as direct delivery within the CNSby intrathecal delivery can be used with, for example, an Omayareservoir. U.S. Pat. No. 5,455,044 provides for the use of a dispersionsystem for CNS delivery [for description of other CNS deliverymechanisms, see U.S. Pat. No. 5,558,852, Betz et al., ibid., andGoldstein and Betz, ibid.]. Tavitan et al. [Tavitan et al. (1998) NatMed 4(4): 467-71] observed that 2′-O-methyl oligonucleotides are able topenetrate into the brain. Other systems make use of specially designeddrugs that utilize the structure and function of the BBB itself todeliver the drugs, for example by designing lipid soluble drugs or bycoupling to peptides that can penetrate the BBB.

It has been shown that stress affects the permeability of the BBB[Sharma H. S. et al. (1992) Prog. Brain Res. 91, 189-196; Ben-Nathan D.et al. (1991) Life. Sci. 489, 1493-1500]. Further, in mammals, acutestress elicits a rapid, transient increase in released acetylcholinewith a corresponding phase of increased neuronal excitability [ImperatoA. et al. (1991) Brain Res. 538, 111-117]. It has been previouslyobserved by the present inventors that the AChE-R isoform and the 14peptide of AChE can act as stress mimicking agents and rupture the BBB.These findings formed the basis for PCT application WO98/22132, thecontents of which are fully incorporated herein by reference. WO98/22132relates to compositions for facilitating the passage of compoundsthrough the BBB, comprising the AChE-R splice variant and/or the peptideI4.

In search for an antisense oligonucleotide targeted against a domain ofthe human AChE, which may be particularly acceptable in human therapy,the inventors have now found, and this is an object of the presentinvention, that a synthetic antisense oligodeoxynucleotide having thenucleotide sequence: 5′-CTGCCACGTTCTCCTGCACC-3′, herein designated SEQID NO:1, is not only useful in selectively suppressing the production ofthe AChE-R isoform, but also possesses cross-species specificity, whichenables its use in rodent animal models of various diseases and,moreover, remarkably appears to penetrate the BBB, and may thus beuseful in treatment of diseases of the central nervous system, alone orin combination with other therapeutic agents. The finding that the novelantisense of the invention can penetrate the BBB was unexpected,particularly in view of the expectation that the BBB would beimpermeable to large polar molecules.

The application of antisense technology to the treatment of nervoussystem disorders has, until recently, been considered to be limited bythe lack of adequate systems for delivering oligonucleotides to thebrain. Nevertheless, several attempts have been made to circumvent thisdifficulty [reviewed in Seidman S. et al. (1999) Antisense Nucl. AcidDrug Devel, 9, 333-340]. Access of chemical agents circulating in theblood to the interstitial spaces of the brain is restricted by thebiomechanical barrier known as the BBB. The strong anionic character ofthe phosphodiester backbone makes oligonucleotides especially poor atcrossing the BBB. In vivo pharmacokinetic studies have demonstrated thatless than 0.01% of a systemically injected dose of a phosphorothioateantisense oligonucleotide may reach the brain, where its residence timemay be as little as 60 min. A research solution to this problem in thelaboratory is direct bypass of the BBB by intracranial injection ofoligonucleotides. Using published stereotactic coordinates for both ratsand mice, oligonucleotides can be delivered by single injections, byrepeated administration through an implanted cannula, or by continuousinfusion using an osmotic mini-pump such as Alzet (Alza, Palo Alto,Calif.). Oligonucleotides can either be delivered into the CSF ordirectly into the brain region of interest. In general, oligonucleotidesare considered to remain relatively localized following intraparenchymaladministration. Thus, a single injection of 24 μg of an antisenseoligonucleotide targeted to the cAMP-response element (CREB) into ratamygdala was reported to diffuse only 0.72±0.04 μl around the injectionsite, exerting region-specific effects on conditioned taste aversion(CTA). Injection of the same oligonucleotide into the basal ganglia 2 mmabove the amygdala had no effect on CTA. Similarly, specific effects onbehavior were reported following the injection of antisenseoligonucleotides against the stress-associated transcription factorc-fos into the medial frontal cortex (single administration; 10 μg),following delivery of oligonucleotides against theneurotransmitter-synthesizing enzyme glutamate decarboxylase into theventromedial hypothalamus (single administration; 1 μg), and following 5days continuous infusion of oligonucleotides targeted to mRNA encodingthe cAMP-responsive transcription factor CREB into the locus coeruleus(20 μg/day). It was further reported that wide distribution ofoligonucleotides in the brain (up to 443 μl around the site of injectionafter 48 hrs) could be achieved by direct, high-flow intraparenchymalmicroinfusion. In that case, the average tissue concentration ofoligonucleotide was calculated to be between 3-15 μM—well within what isconsidered physiologically significant. Regarding uptake into neurons,it was shown that neurons in the striatum of rats preferentially take upoligonucleotides compared to glia. Despite the general retention ofoligonucleotides around the injection site reported in that study, somesignal was observed to be transported along projection pathways todistant sites. However, to be effective therapeutically,oligonucleotides should be prepared in a way that would enable theirstability and free penetrance into the central nervous system followingintravenous injection, or yet more preferably, following oraladministration. Thus, the present invention is aimed at a novel,preferably nuclease protected antisense oligodeoxynucleotide targeted tothe common coding domain of human AChE, which selectively suppresses theproduction of AChE-R, with rapid and long-lasting clinical improvementsin muscle function, which possesses cross-species specificity and canpenetrate the BBB and destroy AChE-R mRNA within central nervous systemneurons.

SUMMARY OF THE INVENTION

The invention relates to a pharmaceutical or medical composition for thetreatment and/or prevention of a progressive neuromuscular disorder,comprising as active ingredient a synthetic antisenseoligodeoxynucleotide targeted against human AChE mRNA having thenucleotide sequence:

5′ CTGCCACGTTCTCCTGCACC 3′. (SEQ ID NO: 1)

The antisense oligonucleotide preferably causes preferential destructionof AChE-R mRNA, possesses cross-species specificity, was demonstrated tocause no toxicity in rodents or primates, and can penetrate the BBB inprimates (monkeys) via both i.v. and p.o. administration routes.

In a preferred embodiment, the synthetic antisense oligodeoxynucleotidehaving the nucleotide sequence designated SEQ ID NO:1 is nucleaseresistant. The nuclease resistance may be achieved by modifying theantisense oligodeoxynucleotide of the invention so that it comprisespartially unsaturated aliphatic hydrocarbon chain and one or more polaror charged groups including carboxylic acid groups, ester groups, andalcohol groups.

In particular embodiments, the nuclease resistant antisenseoligodeoxynucleotide of the invention has at least one of the last three3′-terminus nucleotides is 2′-O-methylated, preferably the last three3′-terminus nucleotides are 2′-O-methylated. Alternatively, the nucleaseresistant antisense oligodeoxynucleotide of the invention may have atleast one of the last 3′-terminus nucleotides fluoridated. Stillalternatively, the nuclease resistant antisense oligodeoxynucleotide ofthe invention has phosphorothioate bonds linking between at least two ofthe last 3′-terminus nucleotide bases, preferably has phosphorothioatebonds linking between the last four 3′-terminal nucleotide bases. Stillalternatively, nuclease resistance may be achieved by the syntheticnuclease resistant antisense oligodeoxynucleotide of the inventionhaving a nucleotide loop forming sequence at the 3′-terminus, forexample a 9-nucleotide loop having the nucleotide sequence CGCGAAGCG(SEQ ID NO:2).

The synthetic nuclease resistant antisense oligodeoxynucleotide of theinvention is capable of selectively modulating mammalian AChEproduction, particularly selectively modulating primate AChE productionin neurons residing in the central nervous system, including human AChEof interneurons.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising an antisense oligodeoxynucleotide of theinvention, and optionally further comprising pharmaceutically acceptableadjuvant, carrier or diluent.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises an antisense oligodeoxynucleotide of SEQ ID NO:1,which is 2′-O-methylated on at least one, preferably the three last3′-terminus nucleotides.

The pharmaceutical composition of the invention is useful in thetreatment and/or prevention of a progressive neuromuscular disorder, forimproving stamina and/or for use in decreasing chronic muscle fatigue.

The pharmaceutical composition of the invention may be for a once dailyuse by a patient of a dosage between about 0.001 μg/g and about 50 μg/gof active ingredient, preferably a dosage of active ingredient of about0.01 to about 5.0 g/g, more preferably a dosage of active ingredient ofabout 0.15 to about 0.5 μg/g.

The pharmaceutical composition of the invention is particularly intendedfor use in treating or preventing a progressive neuromuscular disorder,wherein said disorder is associated with an excess of AChE mRNA orprotein. Such a disorder may be, for example, a progressiveneuromuscular disorder, wherein said disorder is associated with anexcess of AChE-R mRNA.

The pharmaceutical composition of the invention is thus particularlysuitable for treating or preventing a progressive neuromusculardisorder, wherein said disorder is associated with impairment ofcholinergic transmission.

Of particular interest are pharmaceutical compositions for the treatmentof a progressive neuromuscular disorder, wherein said disorder involvesmuscle distortion, muscle re-innervation or NMJ abnormalities, forexample myasthenia gravis, Eaton-Lambert disease, muscular dystrophy,amyotrophic lateral sclerosis, post-traumatic stress disorder (PTSD),multiple sclerosis, dystonia, post-stroke sclerosis, post-injury muscledamage, post-surgery paralysis, excessive re-innervation, andpost-exposure to AChE inhibitors.

The pharmaceutical composition of the invention is also useful inimproving stamina in physical exercise or in decreasing muscle fatigue.

In addition, the invention relates to a pharmaceutical compositioncomprising an antisense oligodeoxynucleotide as denoted by SEQ ID NO:1,for facilitating passage of compounds through the BBB, optionallyfurther comprising additional pharmaceutically active agent and/orpharmaceutically acceptable adjuvant, carrier or diluent. The additionalpharmaceutically active agent is a compound to be transported throughthe BBB, wherein said compound may be contrast agents used for centralnervous system imaging, agents that function to block the effects ofabused drugs, antibiotics, chemotherapeutic drugs and vectors to be usedin gene therapy. This composition would function primarily bysuppressing the production of AChE-R, which is apparently involved inBBB maintenance.

The invention will be described in more detail in the following detaileddescription and on hand of the following figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Human AChE mRNA [GenBank Accession No. M55040; Soreq et al.,Proc. Natl. Acad. Sci. USA 87(24), 9688-9692 (1990)], and human EN 101(hEN101, SEQ ID NO:1), targeted at nucleotides 795-5′ to 3′-814 (shaded)of the coding sequence.

FIG. 2A-B Representation of various physical and chemical properties ofthe human EN101 (SEQ ID NO:1).

FIG. 2A: Internal structure that is expected for the oligonucleotide,and an estimate of the energy (in kcal/mol) required to disrupt thatstructure.

FIG. 2B: Base composition and the predicted melting temperature of itshybrid with the complementary mRNA.

FIG. 3 Mouse AChE mRNA [GenBank Accession No. X56518; Rachinsky et al.(1990) Neuron 5(3), 317-327], and mouse EN101 (mEN101, SEQ ID NO:3),targeted at nucleotides 639-5′ to 3′-658 (shaded) of the codingsequence.

FIG. 4A-B Representation of various physical and chemical properties ofthe mouse EN101 (SEQ ID NO:3).

FIG. 4A: Internal structure that is expected for the oligonucleotide,and an estimate of the energy (in kcal/mol) required to disrupt thatstructure.

FIG. 4B: Base composition and the predicted melting temperature of itshybrid with the complementary mRNA.

FIG. 5 Rat AChE mRNA (partial, 2066 nucleotides) [GenBank Accession No.S50879; Legay et al. (1993), J. Neurochem. 60(1), 337-346], and the ratEN102 (rEN102, SEQ ID NO:5), targeted at nucleotides 51-5′ to 3′-70(shaded) and rat EN101 (rEN101, SEQ ID NO:4), targeted at nucleotides639-5′ to 3′-658 (shaded) of the coding sequence.

FIG. 6A-B Representation of various physical and chemical properties ofthe rat EN101 (SEQ ID NO:4).

FIG. 6A: Internal structure that is expected for the oligonucleotide,and an estimate of the energy (in kcal/mol) required to disrupt thatstructure.

FIG. 6B: Base composition and the predicted melting temperature of itshybrid with the complementary mRNA.

FIG. 7 Immunoreactive AChE-R in EAMG rats.

Separation of rat serum was performed in non-denaturing polyacrylamidegel, and the gel tested for immunoreactive AChE-R. An EAMG rat hadconsiderably higher level of the rapidly migrating rR variant than acontrol rat.

Abbreviations: electroph., electrophoresis; cont., control.

FIG. 8A-C Excess AChE-R expression in muscles of EAMG rats.

FIG. 8A: AChE mRNA transcripts expressed in muscle. Shown is thestress-responding mammalian ACHE gene, with a functional glucocorticoidresponse element (GRE) in its distal enhancer, and its two mRNAtranscripts expressed in muscle. Note that exon 6 is unique to thesynaptic transcript AChE-S, whereas, pseudo-intron 4′ is expressed onlyin the stress induced AChE-R mRNA.

Antibodies targeted to the pseudo-intron 4′-derived C-terminal peptideserved to detect the AChE-R protein, and cRNA probes to exon 6 andpseudo-intron 4′ label the two transcripts (asterisks).

FIG. 8B: Depleted nAChR and excess AChE-R in EAMG muscles. Shown isimmunohistochemical staining of paraffin-embedded sections of tricepsmuscle from normal or EAMG rats treated with the inert inverse(r-invEN102) oligonucleotides, similar to those of untreated rats.Staining was with polyclonal rabbit antibodies to nAChR (1,2) and AChE-R(3,4). Immunopositive areas are stained red. Note that the AChE-Rprotein was prominently elevated and nAChR dramatically reduced in EAMG.In situ hybridization with probes specific for AChE-R or -S mRNAsyielded red stained RNA, with DAPI (white) used to visualize cellnuclei. Note the prominent sub-nuclear accumulation of AChE-R mRNA inpreparations from EAMG, but not control animals (5,6). AChE-S mRNAdisplayed punctuated expression in subnuclear areas in both control andEAMG rats (7,8).

FIG. 8C: rEN101 treatment. In EAMG rats, EN101 reduced levels of AChE-R(1,2) and AChE-R mRNA (5,6), but did not affect nAChR (3,4) or AChE-SmRNA (7,8), as compared to rats treated with the inverse sequence (seeFIG. 8B, above.).

Abbreviations: healt., healthy; r., rat; prot., protein.

FIG. 9A-D Normalized EAMG muscle electrophysiology under suppression ofAChE-R.

FIG. 9A: Immunoreactive AChE-R was detected, as in FIG. 7B, in the serumof healthy and severely affected EAMG rats, treated with rEN101 orr-invEN102, and the densities of the bands are represented in the bargraph.

FIG. 9B: Animals (at least 6 rats in each group) were treated with asingle i.p. injection (75 μg/kg) of the AChE inhibitor neostigmine, andthe CMAP ratio relative to the baseline was measured. The average CMAPratio of EAMG rats included in the study prior to treatment was 87±2.5%of first depolarization, and average CMAP ratio in rEN101-treatedanimals was 107.4±3.8% (inset).

FIG. 9C: Animals (at least 6 rats in each group) were treated withvarious doses of rEN101. The treatment (doses between 10-500 μg/Kg)restored the CMAP decline for up to 72 h. Note that higher dosesconferred increasingly longer-lasting relief.

FIG. 9D: Dose response curse. CMAP responses at each time were plottedas a function of EN101 concentration. Note that at 1 and 5 h there areclearly two effects, a steep increase dependent on a low EN101concentration (IC₅₀<10 μg/kg), superimposed on a much lower-affinityeffect that persists much longer.

Abbreviations: ser., serum; t., time; dep., dependence; neostig.,neostigmine; resp., response; h., hours; perc., percent; cont., control;rat., ratio; bas., baseline.

FIG. 10 Rat EN101 (SEQ ID NO:4) improves stamina in myasthenic rats.

Experimental autoimmune myasthenic gravis (EAMG) rats with varyingseverity of clinical symptoms and healthy Lewis rats were prodded to runon an electrically powered treadmill (25 m/min, inset) until visiblyfatigued. Presented is the average time (sec.±SEM) rats were able to runbefore and 24 h following i.v. administration of 250 μg/kg rEN101. Notethat running time for EAMG rats decreased with disease severity, andincreased for each group treated with rEN101.

Abbreviations: trml., treadmill; treat., treatment; h., hour; clin.,clinical; stat., status; run., running; t., time; sec., seconds.

FIG. 11A-B Stable reversal of declining CMAP response in EAMG ratstreated orally with rEN101.

EAMG rats received rEN101 once daily for up to 4 days by intravenousinjection (25 μg/kg) or via oral gavage (50 μg/kg), or pyridostigmine(1000 μg/kg) by oral gavage. The CMAP ratio was determined 1 and 5 hfollowing the first drug administration and then every 24 h, prior tothe administration of the subsequent dose.

FIG. 11A: Single dose. Orally administered pyridostigmine (n=4) andrEN101 (n=8) relieved the declining CMAP responses within 1 h. 24 hfollowing administration of pyridostigmine, CMAP ratios in muscles oftreated rats returned to the declining baseline. In contrast, no declinewas detected in rats treated with rEN101.

FIG. 11B: Repeated daily doses. The graph depicts the equivalentimprovement in muscle function elicited by oral (50 μg/kg, n=8) ascompared to i.v. (25 μg/kg, n=4) administration of rEN101. Note thatrepeated administration of rEN101 conferred stable, long-termalleviation of CMAP declines. Repeated daily administration ofpyridostigmine at 24 h intervals yielded considerably shorter CMAPimprovements than those obtained with EN101, decreasing back to thedeclining baseline prior to the next dose.

Abbreviations: sing., single; dos., dose; rep., repeated; d., daily;pyridostig., pyridostigmine; h., hours; rat., ratio; bas., baseline;ab., above.

FIG. 12A-C: Long-term rEN101 treatment changes the course of EAMG.

FIG. 12A: Survival. A greater fraction of animals treated once dailywith rEN101 (50 μg/Kg, daily, p.o.) survived than those treated withpyridostigmine (1000 μg/Kg) despite their similarly poor initial statusand initial number of animals in each group.

FIG. 12B: Clinical status. Shown are average values for the clinicalstatus (as defined in Experimental Procedures) of surviving animals fromeach of the treated groups. Note increasing severity of disease insaline- and pyridostigmine-treated animals, as compared to the improvedstatus of rEN 101-treated animals.

FIG. 12C: Stamina. Shown are average running times in sec. for rEN101-and pyridostigmine-treated animals. Note that before treatment, EAMGrats performed as severely sick animals (clinical status 4).

Abbreviations: surv., survival; clin., clinical; stat., status; stam.,stamina; an., animals; al., alive; sc., score; run., running; t., time;sec., seconds; w., weeks; sal., saline; pyridostig., pyridostigmine.

FIG. 13 Proposed model for EN101 activity

At the neuromuscular junction, acetylcholine (ACh) released from themotoneuron terminal (top) into the synaptic cleft travels towards themuscle postsynaptic membrane (below). There, it interacts with nAChR toinitiate an inward ion current and elicit muscle action potentials. AChis subsequently hydrolyzed by synapse-bound AChE-S. Subsynaptic musclenuclei (ellipses) produce, in addition to the primary AChE-S mRNAtranscript, the normally rare AChE-R mRNA with its alternative 3′-end.This transcript translates into soluble, secretory AChE-R monomers.Myasthenic autoimmune antibodies toward nAChR block the initiation ofaction potentials, mimicking an ACh-deficient state. The cholinergicimbalance results in AChE-R accumulation that enhances ACh destruction,leading to muscle fatigue. Chemical anticholinesterases (indentedcircles) non-selectively block both AChE-S and AChE-R, which transientlyincreases ACh levels, yet further intensifies AChE-R overproduction. Incontrast, the antisense agent EN101 selectively induces AChE-R mRNAdestruction, preventing AChE-R synthesis while maintaining AChE-S andsustaining normal neuromuscular transmission.

FIG. 14 Dose-dependent hEN101 suppression of neuronal AChE-R mRNA, butnot of AChE-S mRNA.

Shown are representative fields from spinal cord sections ofhEN101-treated monkeys following in situ hybridization with AChE-R orAChE-S cRNA probes. Note that AChE-R mRNA labeling decreased, but AChE-SmRNA levels appeared unchanged. An increasing dose of o.g.-administeredhEN101 suppressed AChE-R mRNA more effectively, suggestingdose-dependence. Administration of the higher dose via i.v. appearedmore effective than the o.g. route.

Abbreviations: d., day.

FIG. 15 hEN101-suppression of neuronal AChE-R mRNA levels is celltype-specific.

Spinal cord neurons from hematoxylin-eosin stained monkey sections weredivided by size into cells with perikaryal diameters of <40, 40 to 70and >70 μm. The percent of cells within each size group that werepositively labeled for AChE-R mRNA was recorded in 5 different fields of1 mm² each, for each hEN101 treatment. Note that hEN101 effectivenesswas apparently highest in the relatively small interneurons, and lowestin the largest motoneurons.

Abbreviations: pos., positive; cel., cell; siz., size; gr., group; bo.,body; diam., diameter; nv., naïve.

FIG. 16 Shown are levels of hydrolyzed acetylthiocholine, indicatingacetycholinesterase activity in the plasma of cynomolgous monkeystreated i.v. for two consecutive days with 150 or 500 μg/kg hEN101 orwith orally administered 500 μg/kg hEN 101.

FIG. 16A: Total activity.

FIG. 16B: Activity under 5×10⁻⁵ M of iso-OMPA (AChE). Noteinjection-induced increases in enzyme activity and AS-ON reductions.

Abbreviations: t., time; fol., following; treat., treatment; hrs.,hours.

FIG. 17 Effect of rEN101 on AChE-R mRNA in rat spinal cord neurons. Thepresence of AChE-R mRNA-positive cells was determined in spinal cordsection of rats that had been treated for 7 days with rEN101 (500 μg/kg,i.v., daily).

Abbreviations: cont., control.

FIG. 18 hEN101 alleviates ptosis in MG patients. Photographs show:before hEN101 treatment (upper panel), on 10 Mestinon®/day, 600 mg;during hEN101 treatment (middle panel), 500 μg/kg, 2-day treatment; and4 weeks after hEN101 treatment (lower panel), back to Mestinon®treatment.

FIG. 19 Efficacy of oral hEN101 in MG patients. Graph shows mean changefrom baseline of one patient in total grade. This patient was a 56 yearold male, myasthenic for 29 years, who was treated with pyridostigmineand had a baseline of QMG=6. He returned to the pyridostigmine treatment72 hours after the last hEN101 dose.

FIG. 20 Improvement in Total QMG Score. Graph shows the mean percentageimprovement (plus standard deviation) in total QMG score of allpatients, from the baseline value until day 6 of treatment.

FIG. 21A-B hEN101 improves myasthenic status.

FIG. 21A Graph shows QMG score of all the patients included in theclinical trial.

FIG. 21B Graph shows mean change from baseline in QMG score.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of clarity, the following abbreviations and terms aredefined herein:

-   -   AChE: acetylcholinesterase    -   AChE-R: acetylcholinesterase, “readthrough” variant or isoform,        its mRNA includes pseudo-intron 14    -   AChE-S: acetylcholinesterase, synaptic variant or isoform    -   AS-ON: antisense oligonucleotide    -   BBB: blood-brain barrier    -   CMAP: compound muscle action potential    -   CNS: central nervous system    -   EAMG rat: rats wherein experimental autoimmune myasthenia gravis        has been induced    -   EN101: may also be referred as AS3; antisense oligonucleotide        targeted against human, rat or mouse (hEN101, rEN101 or mEN101,        respectively) AChE mRNA    -   EN102: may also be referred as -AS1, antisense oligonucleotide        targeted against AChE mRNA, at a different region than EN101    -   MG: myasthenia gravis, a neuromuscular junction disease    -   i.v.: intravenous    -   o.g.: oral gavage    -   p.o.: per os        Antisense oligonucleotide: A nucleotide comprising essentially a        reverse complementary sequence to a sequence of AChE mRNA. The        nucleotide is preferably an oligodeoxynucleotide, but also        ribonucleotides or nucleotide analogues, or mixtures thereof,        are contemplated by the invention. The antisense oligonucleotide        may be modified in order to enhance the nuclease resistance        thereof, to improve its membrane crossing capability, or both.        The antisense oligonucleotide may be linear, or may comprise a        secondary structure. It may also comprise enzymatic activity,        such as ribozyme activity.

Progressive neuromuscular disorder: A disorder or condition associatedwith excess AChE mRNA or protein production, characterized by changes inthe morphology of the NMJ and impairment in neuromuscular transmission.The neuromuscular disorder may involve muscle distortion, musclere-innervation or NMJ abnormalities. More preferably, the progressiveneuromuscular disorder is myasthenia gravis, muscular dystrophy,multiple sclerosis, amyotrophic lateral sclerosis, post-traumatic stressdisorder (PTSD), or dystonia.

The present invention relates to a novel antisense oligodeoxynucleotidesubstantially as denoted by SEQ ID NO:1, also designated herein ashEN101.

In addition to the part of the sequence which is complementary to AChEsequence, the antisense oligonucleotide of the invention may alsocomprise RNA sequences with enzymatic nucleolytic activity, or may belinked to such sequences. Preferred nucleolytic sequences are ribozymesequences, which were shown to specifically interact with mRNAtranscripts. They are ribonucleic acid sequences, including RNase activesites flanked by antisense oligonucleotides [Haseloff and Gerlach (1988)Nature 3, p. 585, Sarver et al. (1990) Science 247, p. 1222]. Preferredribozymes are hammerhead ribozymes [Conaty et al. (1999) Nucleic AcidsRes. 27, 2400-2407; and Xu et al. (1999) Endocrinology, 140, 2134-44].Another preferred ribozyme is the hairpin ribozyme structure, e.g., asderived from tobacco ringspot virus satellite RNA [see Perez-Ruiz (1999)Antisense Nucleic Acid Drug Dev., 9, 33-42].

The novel antisense oligodeoxynucleotide of the invention corresponds tothe reverse complement of human AChE mRNA sequence, from nucleotide795-5′ to nucleotide 3′-814 (FIG. 1). Prior work by the presentinventors has demonstrated the usefulness of antisense oligonucleotidein suppressing AChE production and in the treatment of memorydeficiency. In said prior work, a number of AChE antisenseoligonucleotides have been disclosed. Said prior work further disclosesdesirable features of such antisense oligonucleotides and possiblemodifications thereof, such as nuclease resistance, modifications toenhance membrane transport of oligonucleotides, and the like. Said priorwork, e.g. WO 98/26026, is therefore incorporated herein in its entiretyby reference. In another publication, the present inventors describe therole of antisense oligonucleotides in the treatment of a variety ofneurodegenerative diseases [Seidman, S. et al., Antisense Res. Nucl.Acids Drug Devel. 9, 333-340 (1999)].

The antisense oligodeoxynucleotide of the invention is preferablynuclease resistant. There are a number of modifications that impartnuclease resistance to a given oligonucleotide. Reference is made to WO98/26062, which publication discloses that oligonucleotides may be madenuclease resistant e.g., by replacing phosphodiester internucleotidebonds with phosphorothioate bonds, replacing the 2′-hydroxy group of oneor more nucleotides by 2′-O-methyl groups, or adding a nucleotidesequence capable of forming a loop structure under physiologicalconditions to the 3′ end of the antisense oligonucleotide sequence. Anexample for a loop forming structure is the sequence 5′ CGCGAAGCG (SEQID NO:2), which may be added to the 3′ end of a given antisenseoligonucleotide to impart nuclease resistance thereon.

The cells on which the antisense oligonucleotide of the invention exertsits effects are preferably muscle cells and cells of the NMJ, includingthe nerve axons and endplate structures.

Using the antisense oligonucleotides according to the invention, it isexpected that AChE-R amount and AChE-R mRNA levels are reduced incentral nervous system neurons by at least about 30%, preferably by atleast about 40%, and more preferably by at least about 50%, within 24 hrof the treatment, and by about 80% under repeated treatment. Thisreduction was shown by fluorescent in situ hybridization (FISH) andimmune labeling and its effectiveness was confirmed by electrophysiologyand tread mill tests. It exceeded by far all previous reports of AS-ONdestruction of AChE-R mRNA in other cells and tissues.

In yet another embodiment of the invention, the preferred treatmentwindow of candidate oligonucleotides is evaluated by FISH. The techniqueof in situ hybridization is well known to the man of skill in the art,and is described e.g., In situ Hybridization, Wilkinson, D. G. (Ed.)ISBN: 0199633274; In situ Hybridization for the Brain, Wisden W., MorrisB. J. (Eds.), ISBN: 0127599207, PCR in situ Hybridization: A PracticalApproach (Practical Approach Series 186), Herrington C. S., John O'LearyJ., (Eds.) ISBN:019963632X. Detailed protocols relating to in situhybridization using non-radioactively labeled probes are available fromMicrosynth GmbH (Balgach, Switzerland).

Labeled AChE-R cRNA sequences may be used as probes for in situhybridization. The ACHE cRNA probe preferably comprises I4 pseudo-intronsequences.

In a preferred embodiment of the invention, the AChE mRNA determinationis carried out by using in situ RT-PCR, which technique is described,e.g., in the above-mentioned references, see also PCR in situhybridization: Protocols and Applications, 3rd ed., by Nuovo, G. J.Lippincott, Raven Press, New York (1996).

Phosphorothioate-modified oligonucleotides are generally regarded assafe and free of side effects. Peng et al. teach that undesired in vivoside effects of phosphorothioate antisense oligonucleotides may bereduced when using a mixed phosphodiester-phosphorothioate backbone. Theantisense oligonucleotides of the present invention have been found tobe effective as partially phosphorothioates and yet more effective aspartially 2′-O-methyl protected oligonucleotides. WO 98/26062 teachesthat AChE antisense oligonucleotides containing three phosphorothioatebonds out of about twenty internucleotide bonds are generally safe touse in concentrations of between about 1 and 10 μM. However, forlong-term applications, oligonucleotides that do not release toxicgroups when degraded may be preferred. These include 2′-O-methylprotected oligonucleotides, but not phosphorothioate oligonucleotides. Afurther advantage of 2′-O-methyl protection over phosphorothioateprotection is the reduced amount of oligonucleotide that is required forAChE suppression. This difference is thought to be related to theimproved stability of the duplexes obtained when the 2′-O-methylprotected oligonucleotides are used [Lesnik, E. A. & Freier, S. M.,Biochemistry 37, 6991-7, (1998)]. An alternative explanation for thegreater potency of the 2′-O-methyl oligonucleotides is that thismodification may facilitate penetration of the oligonucleotide chainthrough the cell membrane. A further advantage of 2′-O-methyl protectionis the better protection against nuclease-mediated degradation that itconfers, thus extending the useful life time of antisenseoligonucleotides protected in this way.

In accordance with the invention, the dosage of the antisenseoligodeoxynucleotide is about 0.001 to 50 μg oligonucleotide per gram ofbody weight of the treated animal. Preferably, the dosage is about 0.01to about 5.0 μg/g. More preferably, the dosage is between about 0.05 toabout 0.7 μg/g. Thus, the optimal dose range is between 50-500 g/kg ofbody weight of the treated subject, for rats, monkeys and also humans.

The antisense oligonucleotide of the invention is provided for use inthe treatment of a disorder that involves excessive AChE mRNAproduction.

The disorder is preferably a disorder involving functional andmorphological changes in the NMJ.

The progressive neuromuscular disorder preferably involvesoverexpression of AChE-R mRNA.

More preferably, the disorder is selected from, but not limited to,multiple sclerosis, PTSD, myasthenia gravis, muscular dystrophy,amyotrophic lateral sclerosis, dystonia, muscle distortion, musclere-innervation or excessive muscle innervation.

The excessive muscle innervation is selected preferably from, but notlimited to, excessive innervation after trauma, preferably afteramputation.

In one aspect, the invention relates to a pharmaceutical composition forthe treatment and/or prevention of a progressive neuromuscular disorder,for improving stamina in physical exercise and/or for use in decreasingchronic muscle fatigue, comprising as active ingredient the syntheticantisense oligodeoxynucleotide hEN101, as denoted by SEQ ID NO:1, andoptionally further comprising additional therapeutic agents and/orpharmaceutically acceptable carriers, excipients and/or diluents.Preferably, said pharmaceutical composition is for the treatment and/orprevention of myasthenia gravis.

The progressive neuromuscular disorder to be treated and/or prevented bythe pharmaceutical composition of the invention is associated with anexcess of AChE or protein. Usually, said excessive AChE will be theAChE-R variant or isoform.

In addition, said progressive neuromuscular disorder to be treatedand/or prevented by the pharmaceutical composition of the invention isassociated with impairment of the cholinergic transmission. Saiddisorder may involve muscle distortion, muscle re-innervation, orneuromuscular junction (NMJ) abnormalities.

The pharmaceutical composition of the invention is for use in thetreatment and/or prevention of a disorder such as myasthenia gravis(MG), Eaton-Lambert disease, muscular dystrophy, amyotrophic lateralsclerosis (ALS), post-traumatic stress disorder (PTSD), multiplesclerosis (MS), dystonia, post-stroke sclerosis, post-injury muscledamage, excessive re-innervation, post-surgery paralysis of unknownorigin and post-exposure to AChE inhibitors.

In one embodiment, the pharmaceutical composition of the invention isfor daily use by a patient in need of such treatment, at a dosage ofactive ingredient between about 0.001 μg/g and about 50 μg/g.Preferably, the treatment and/or prevention comprises administering adosage of active ingredient of about 0.01 to about 5.0 μg/g. Mostpreferably, said dosage of active ingredient is of between about 0.05 toabout 0.70 μg/g, and even most preferably, the dosage is from 0.15 to0.50 μg/g of body weight of the patient.

As may be seen in Example 11 and FIGS. 18-21, treatment of MG patientswith hEN101 resulted in significant improvement of the clinicalsymptoms. As an example of such improvements, FIG. 18 shows how patientswere capable of better opening their eyes (lifting their eye lids), andtheir QMG scores improved significantly (FIG. 19-21). Thus, hEN101 hasproved to be effective in reversing the symptoms in human MG patients.

The pharmaceutical composition of the invention may optionally compriseat least one additional active agent. Said active agent may be, forexample, AChE inhibitors used for the treatment of neuromusculardisorders.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising an antisense oligodeoxynucleotide as denoted bySEQ ID NO:1, for facilitating passage of compounds through the BBB,optionally further comprising additional pharmaceutically active agentand/or pharmaceutically acceptable adjuvant, carrier or diluent. Theadditional pharmaceutically active agent is a compound to be transportedthrough the BBB. These pharmaceutical compositions of the invention maybe used for treatment of disorders associated with the central nervoussystem, particularly such disorders that require administration of anactive agent into the CNS, for example, for the treatment of braintumors. Conventional chemotherapeutic agents do not pass the BBB, andare therefore ineffective [de Angelis, L. M., N. Engl. J. Med. 433,114-123 (2001)]. As the antisense oligonucleotide of the invention hasbeen shown to penetrate the BBB, brain tumors could be treated byinjection or oral administration of the antisense oligonucleotide of theinvention, preventing or reducing the need for methods requiringinvasion of the CNS. Antisense oligonucleotides can be madetumor-specific [Ratajczak M. Z. et al. Proc. Natl. Acad. Sci. USA 89,11823-11827 (1992)]; therefore should they be found to pass the BBB,they may be both specific and effective. Thus, the additionalpharmaceutical agents comprised in these compositions of the inventionmay be, for example carcinostatic and metastatic drugs.

A number of compounds are needed for the diagnostic or treatment ofconditions affecting the central nervous system, wherein the BBB wouldnormally impede their delivery. These conditions can include any diseaseor pathology, which include but are not limited to infections,neurochemical disorders, brain tumors and gliomas, demyelination, otherneuropathies, encephlopathies, coma, ischemia, hypoxia, epilepsy,dementias, cognitive disorders, neuropsychiatric disorders (as forexample depression, anxiety, schizofrenia and the like), as well asgenetic disorders. Thus, said compounds or additional pharmaceuticallyactive agent to be transported across the BBB may be, for example,contrast agents (dyes) used for central nervous system imaging, drugssuch as antibiotics or chemotherapeutics, gene therapy vectors, or evenagents that function to block the effects of abused drugs. Theadministration and dosage of these compounds shall be according to whatis known in medical practice, which generally should take into accountthe clinical condition of the patient in need of such treatment, as wellas said patient's age, sex, body weight and other factors known to beimportant in the medical practice. The site and method of administrationshould also be chosen accordingly. The pharmaceutically effective amountfor purposes herein is thus determined by such considerations as areknown in the art. The compound can be administered in several ways asdescribed for the delivery of the composition (see below).

The compound to be transported through the BBB may be administeredsimultaneously with the composition of the invention or can beadministered at some point during the biologically effective period ofthe action of the composition. In other words, the composition of theinvention facilitates the disruption of the BBB, i.e. it opens the BBB,for a period of time depending on its dose and the compound can then beadministered during this “open” period.

In order to be effective, the antisense oligonucleotide of theinvention, also when comprised in a pharmaceutical composition of theinvention, must travel across cell membranes. In general, antisenseoligonucleotides have the ability to cross cell membranes, apparently bya saturable uptake mechanism linked to specific receptors. As antisenseoligonucleotides are single-stranded molecules, they are to a degreehydrophobic, which enhances passive diffusion through membranes.Modifications may be introduced to an antisense oligonucleotide toimprove its ability to cross membranes. For instance, theoligonucleotide molecule may be linked to a group comprising optionallypartially unsaturated aliphatic hydrocarbon chain and one or more polaror charged groups such as carboxylic acid groups, ester groups, andalcohol groups. Alternatively, oligonucleotides may be linked to peptidestructures, which are preferably membranotropic peptides. Such modifiedoligonucleotides penetrate membranes more easily, which is critical fortheir function and may therefore significantly enhance their activity.Palmityl-linked oligonucleotides have been described by Gerster et al.[Anal. Biochem. 262, 177-84 (1998)]. Geraniol-linked oligonucleotideshave been described by Shoji et al. [J. Drug Target 5, 261-73 (1998)].Oligonucleotides linked to peptides, e.g., membranotropic peptides, andtheir preparation have been described by Soukchareun et al. [Bioconjug.Chem. 9, 466-75 (1998)]. Modifications of antisense molecules or otherdrugs that target the molecule to certain cells and enhance uptake ofthe oligonucleotide by said cells are described by Wang, J. [ControlledRelease 53, 39-48 (1998)].

Any of the compositions of the invention are for use by injection,topical administration or oral uptake. Preferred uses of thepharmaceutical compositions of the invention by injection aresubcutaneous injection, intraperitoneal injection, and intramuscularinjection. As shown in the following Examples, oral administrationproved very effective, it is much easier to prescribe and meet patientcompliance, and it involves more easy handling.

The compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,sachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with a binder (e.g., povidone,gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent,preservative, disintegrant (e.g., sodium starch glycolate, cross-linkedpovidone, cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

The pharmaceutical compositions of the invention generally comprise abuffering agent, an agent which adjusts the osmolarity thereof, andoptionally, one or more carriers, excipients and/or additives as knownin the art, e.g., for the purposes of adding flavors, colors,lubrication, or the like to the pharmaceutical composition. A preferredbuffering agent is phosphate-buffered saline solution (PBS), whichsolution is also adjusted for osmolarity.

Carriers may include starch and derivatives thereof, cellulose andderivatives thereof, e.g., microcrystalline cellulose, xantham gum, andthe like. Lubricants may include hydrogenated castor oil and the like.

A preferred pharmaceutical formulation is one lacking a carrier. Suchformulations are preferably used for administration by injection,including intravenous injection.

The preparation of pharmaceutical compositions is well known in the artand has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack PublishingCo., Easton, Pa., 1990, and especially pp. 1521-1712 therein.

Additives may also be designed to enhance uptake of the antisenseoligonucleotide across cell membranes. Such agents are generally agentsthat will enhance cellular uptake of double-stranded DNA molecules. Forinstance, certain lipid molecules have been developed for this purpose,including the transfection reagents DOTAP (Roche Diagnostics),Lipofectin, Lipofectam, and Transfectam, which are availablecommercially. For a comparison of various of these reagents in enhancingantisense oligonucleotide uptake see e.g., Quattrone et al. [Biochemica1, 25, (1995)] and Capaccioli et al. [Biochem. Biophys. Res. Comm. 197,818 (1993). The antisense oligonucleotide of the invention may also beenclosed within liposomes. The preparation and use of liposomes, e.g.,using the above mentioned transfection reagents, is well known in theart. Other methods of obtaining liposomes include the use of Sendaivirus or of other viruses. Examples of publications disclosingoligonucleotide transfer into cells using the liposome technique aree.g., Meyer et al. [J. Biol. Chem. 273, 15621-7 (1998)], Kita and Saito[Int. J. Cancer 80, 553-8 (1999)], Nakamura et al. [Gene Ther. 5,1455-61 (1998)] Abe et al. [Antivir. Chem. Chentother. 9, 253-62(1998)], Soni et al. [Hepatologv, 28, 1402-10 (1998)], Bai et al. [Ann.Thorac. Surg. 66, 814-9 (1998) and see also discussion in the samejournal p. 819-20], Bochot et al. [Pharm. Res. 15, 1364-9 (1998)],Noguchi et al. [FEBS Lett. 433, 169-73 (1998)], Yang et al. [Circ. Res.83, 552-9 (1998)], Kanamaru et al. [J. Drug Target. 5, 235-46 (1998)]and references therein. The use of Lipofectin in liposome-mediatedoligonucleotide uptake is described in Sugawa et al. [J. Neurooncol. 39,237-44 (1998)]. The use of fusogenic cationic-lipid-reconstitutedinfluenza virus envelopes (cationic virosomes) is described in Waelti etal. [Int. J. Cancer, 77, 728-33 (1998)].

The above-mentioned cationic or nonionic lipid agents not only serve toenhance uptake of oligonucleotides into cells, but also improve thestability of oligonucleotides that have been taken up by the cell.

The invention also relates to a method for the treatment or preventionof a progressive neuromuscular disorder or other disease involvingexcessive production of AChE-R mRNA, comprising administering theoligodeoxynucleotide of the invention or a pharmaceutical composition ofthe invention or of any of the preferred embodiments thereof, to apatient in need thereof.

Lastly, the invention relates to a method of administering to a patientin need of such treatment a therapeutic agent for treatment of adisorder or disease of the CNS, comprising the steps of administering tosaid patient the antisense oligodeoxynucleotide of the invention andsaid therapeutic agent. The administration of the therapeutic agent maybe simultaneous with the administration of that of the antisenseoligodeoxynucleotide of the invention, or preceding or following thesame. Rupture of the BBB by the antisense oligodeoxynucleotide of theinvention will facilitate the passage of the therapeutic agent acrossthe BBB and into the CNS, where its effect is required.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Experimental Procedures Animals:

Rats: EAMG was induced in female Lewis rats (120-150 g) purchased fromthe Jackson Laboratory (Bar Harbor, Me.), and housed in the AnimalFacility at the Hebrew University Faculty of Medicine, in accordancewith NIH guidelines. Control FVB/N mice were subjected to confined swimstress as described [Kaufer et al., 1998 id ibid.]. Transgenic FVB/Nmice overexpressing AChE-R were as detailed [Sternfeld et al. (2000)Proc. Natl. Acad. Sci. USA 97, 8647-8652].

Monkeys: Purpose-bred female and male 15 month-old Cynomolgus monkeyswere used.

Oligonucleotides: HPLC-purified, GLP grade oligonucleotides (purity >90%as verified by capillary electrophoresis) were purchased from Hybridon,Inc. (Worchester, USA). Lyophilized oligonucleotides were resuspended insterile double distilled water (24 mg/ml), and stored at −20° C. Theoligonucleotides were prepared with phosphodiester linkages at all butthe three terminal 3′ positions at which 2′-O-methyl ribonucleotidesubstitutions were made. The primary sequences used in this study were:

(human AS3, SEQ ID NO: 1) hEN 101 5′-CTGCCACGTTCTCCTGCACC-3′2′-O-methylated 5′-CTGCCACGTTCTCCTGCA*C*C*-3′ hEN101 (methylatednucleotides marked with *) (mouse AS3, SEQ ID NO: 3) mEN1015′-CTGCAATATTTTCTTGCACC-3′ [Grifman, M., and Soreq. H. (1997) AntisenseNucleic Acid Drug Dev 7, 351-9] (rat AS3, SEQ ID NO: 4) rEN1015′-CTGCGATATTTTCTTGTACC-3′ [WO98/26062] (SEQ ID NO: 5) rEN1025′-GGGAGAGGAGGAGGAAGAGG-3′ [WO98/26062] (SEQ ID NO: 6) r-invEN1025′-GGAGAAGGAGGAGGAGAGGG-3′ [Meshorer, E. et al. (2002) id ibid]

Stability of hEN101: hEN101 was found to be stable (>90% of originalconcentration) after storage for 2 h in human plasma with EDTA at roomtemperature, following three freeze/thawing cycles, or after 1 month at−20° C. Exposure at room temperature to Li-heparin-treated blood causeda decay of hEN101 with a half-life in the order of 30 min.

Antibodies: Rabbit polyclonal antibodies against the C-terminal AChE-Rwere prepared and purified as described [Sternfeld et al. (2000) ibid.].Goat polyclonal anti-AChR (C-20, S.C.-1448) antibodies were from SantaCruz, (Santa Cruz, Calif.).

Induction of EAMG: Torpedo acetylcholine receptor (T-AChR) was purifiedfrom T. californica electroplax by affinity chromatography onneurotoxin-Sepharose resin, as previously described [Boneva, N. et al.(2000) Muscle & Nerve 23, 1204-8]. Rats were immunized with 40 μg ofpurified T-AChR emulsified in complete Freund's adjuvant supplementedwith 1 mg of M. tuberculosis H37Ra (Difco, Detroit Mich.). The animalswere injected subcutaneously in the hind footpads and a boosterinjection of the same amount was given after 30 days. A third injectionwas administered to animals that did not develop EAMG after the secondinjection. Animals were weighed and inspected weekly during the firstmonth and daily after the booster immunization, for evaluation of muscleweakness. The clinical status of the rats was graded according to:0—Without definite weakness (treadmill running time, 23±3 min); mild(1)—weight loss >3% during a week, >10 min. running time on treadmill;moderate (2)—moderate weakness accompanied by weak grip or cry withfatigue, weight loss of 5-10%, 3-5 min. running on treadmill;moderate-severe (3)-moderate to severe weakness, hunched back posture atrest, head down and forelimb digit flexed, tremulous ambulation, 10%body weight loss, 1-2 min. run on treadmill); severe (4)—severe generalweakness, no cry or grip, treadmill running time <1 min, weightloss >10%; (5)—death.

Anti-AChR antibody determination: Serum was assayed by directradioimmunoassay, using ¹²⁵I-α bungarotoxin (BgT) bound to T-AChR and torat (R) AChR [Boneva et al. (2000) id ibid]. All the EAMG rats displayedhigh anti-T-AChR or anti-R-AChR titers, with serum mean±standard error(SE) values of 82.1±16.0 nM for anti-T-AChR antibodies and 19.9±1.8 nMfor anti-R-AChR. Human serum was tested for the level of anti-AChRantibodies as previously described [Drachman, D. B. (1994) N Engl J Med330, 1797-810].

Quantification of nAChR: AChR concentration in the gastrocnemius andtibialis muscles was determined using ¹²⁵I-α-BgT binding followed byprecipitation by saturated ammonium sulfate as described previously[Boneva et al. (2000) id ibid].

Immunocytochemistry: Muscle sections were deparaffinized with xylene andwere re-hydrated in graded ethanol solutions (100%, 90%, 70%) and PBS.Heat-induced antigen retrieval was performed by microwave treatment (850W for rapid boil following 10 min in reduced intensity) in 500 ml of0.01M citrate buffer pH 6.0. Slides were cooled to room temperature andrinsed in double distilled water. Non-specific binding was blocked by 4%normal donkey serum in PBS with 0.3% Triton X-100 and 0.05% Tween 20 (1hr at room temperature). Biotinylated primary antibody was diluted(1:100 and 1:30 for rabbit anti-AChE-R [Sternfeld et al. (2000) id ibid]and goat anti-nAChR, respectively) in the same buffer and slides wereincubated 1 hr at room temperature following overnight incubation at 4°C. Sections were rinsed and incubated with alkalinephosphatase-conjugated secondary antibody, diluted in the same blockingbuffer 1 hr at room temperature and then overnight at 4° C. Detectionwas with the alkaline phosphatase substrate Fast Red (Roche Diagnostics,Mannheim, Germany). Slides were simultaneously transferred to a stopsolution (25 mM EDTA, 0.05% Triton X-100, 1 mM levamisole in PBS, pH7.2), rinsed in PBS and cover-slipped with Immunomount (Shandon).

For spinal cord sections, primary mouse anti-SC35 antibody was diluted(1:100) in the same buffer as the previous primary antibodies, andslides were incubated 1 h at room temperature following overnightincubation at 4° C. Sections were rinsed and incubated with peroxidaseconjugated goat anti-mouse secondary antibody, diluted in the sameblocking buffer, for 1 h at room temperature and then overnight at 4° C.Detection was performed with DAB substrate (Sigma). Slides werecover-slipped with Immunomount (Shandon, Pittsburgh, Pa.).

Electromyography: Rats were anesthetized by i.p. injection of 2.5 mg/Kgpentobarbital, immobilized, and subjected to repetitive sciatic nervestimulation, using a pair of concentric needle electrodes at 3 Hz.Baseline compound muscle action potential (CMAP) was recorded by aconcentric needle electrode placed in the gastrocnemius muscle,following a train of repetitive nerve stimulations at supramaximalintensity. Decrease (percent) in the amplitude of the fifth vs. thefirst muscle action potential was determined in two sets of repetitivestimulations for each animal. A reduction of 10% or more was consideredindicative of neuromuscular transmission dysfunction.

Drug administration: Intravenous injections and blood sampling foranti-AChR antibodies testing were via the right jugular vein underanesthesia. For oral administration, a special needle for oral gavagefeeding was used, which is curved with a ball end (Stoelting, Wood DaleIll.). Mestinon® was administered in a dose of 1 mg/kg/day, andpurchased from Hoffmann La-Roche, Basel, Switzerland.

Exercise training on treadmill: To establish a clinical measure ofneuromuscular performance in EAMG rats, a treadmill assay was performed.Animals were placed on an electrically powered treadmill [Moran et al.(1996) J Therm Biol 21, 171-181] at 25 m/min (a physical effort ofmoderate intensity) until visibly fatigued. The amount of time the ratswere able to run was recorded before and after anti-sense or Mestinon®treatment.

In situ hybridization: Tissues were fixed in 4% paraformaldehyde and cutinto 7 Lm paraffin embedded sections. Spinal cord sections weredeparaffinized, rehydrated using serial ethanol dilutions andpermeabilized with proteinase K (10 μg/ml at room temp.). Slides wereexposed to 5′ biotinylated, fully 2′-oxymethylated AChE-R orAChE-S-specific 50-mer cRNA probes complementary to human ACHEpseudo-intron 4 or exon 6, respectively [Grisaru et al. (2001) idibid.]. Hybridization was performed overnight at 52° C. in hybridizationmixture containing 10 μg/ml probe, 50 μg/ml yeast tRNA, 50 μg/ml heparinand 50% formamide in 375 mM Na chloride, 37.5 mM Na citrate, pH 4.5. Formonkey sections, the probe was constructed according to the human AChE-Rsequence; for rat sections, according to the mouse sequence. Slides werewashed to remove non-hybridized probe, blocked with 1% skim milkcontaining 0.01% Tween-20 and 2 mM levamisole, an alkaline phosphataseinhibitor used to suppress non-specific staining and incubate withstreptavidin-alkaline phosphatase (Amersham Pharmacia). FastRed™substrate (Roche Diagnostics) was used for detection. DAPI staining(Sigma Chemical Co., St. Louis, Mo., USA) served to visualize nuclei.Microscope images were analyzed with Image Pro Plus 4.0 (MediaCybernetics) software.

Serum analyses: Blood samples drawn from EAMG rats and MG patients weresubjected to non-denaturing gel electrophoresis as described [Kaufer etal. (1998) id ibid], as well as to catalytic activity measurements ofAChE [Shapira et al. (2000) id ibid]. Iso-OMPA(tetraisopropylpyrophosphoramide, 5×10-5 μM), was used to blockbutyrylcholinesterase activity in the serum samples. For activitystaining on polyacrylamide gels [Kaufer et al. (1998) id ibid] we used510-6 M iso-OMPA.

Protocol for Phase Ib Clinical Trial of MG patients using hEN101:Patients were hospitalized and pyridostigmine was discontinued for 12-18hours before EN101 testing. Assessment of MG status was performed firstat entry, then after pyridostigmine stoppage, and regularly after EN101treatment using a Quantitative MG (QMG) score. Escalating oral doses ofEN101 (10-150 g/kg) were given in the first day (day 1), followed by adaily dose of 500 μg/kg for 3 days (days 2-4). Days 5 and 6 were washoutperiod without pyridostigmine, and restitution of pyridostigmineoccurred when it became necessary. Patients were monitored for 1 monththereafter, with three visits as out-patients.

The following parameters were used as inclusion criteria:

-   -   Class II and above, according to MGFA classification (myasthenia        gravis standard clinical classification of the disease severity        score);    -   Age 18-70;    -   Seropositive for AChR antibodies;    -   Patients under pyridostigmine (180 mg/day) treatment without        concomitant immunosuppressants;    -   Stable for 3 months, with no PE (plasma exchange) for 6 months;    -   No other major or active diseases.

Evaluation of the MG status of each patient was based on the followingparameters:

-   (a) QMG scoring (maximum value of 9), based on the measurements of:    -   Fatigue in each limb (4);    -   Fatigue of the neck (1);    -   Swallowing rate (1);    -   Power in the hands (2);    -   Respirometry (1).    -   These measurements were taken daily, 4 times per day, and        averaged for days 2-6.-   (b) Patient's subjective report;-   (c) Vital signs, clinical chemistry, hematology, urinalysis, ECG and    physical examination, recorded daily.

Example 1 AChE-R Accumulate in Blood and Muscle of EAMG Rats

As previously shown by the inventors, the AChE-R variant migrates onnon-denaturing polyacrylamide gels faster than the tetrameric synapticenzyme, AChE-S [Kaufer et al. (1998) id ibid.], and it is present in theserum of MG patients [WO01/36627]. Similarly, immunoblot analysisconfirmed that in EAMG rats, as compared with healthy rats, there was amassive increase in serum AChE-R (FIG. 7).

Expression of alternative AChE variants (FIG. 8A), as well as of thenicotinic acetylcholine receptor (nAChR), was tested in control and EAMGrats. Depletion of nAChR in muscle sections from EAMG rats was detected,as evidenced by a quantitative immunoassay using antibodies againstnAChR (FIG. 8B, 1 and 2). The immunostaining showed that muscle nAChRwas reduced by 48±7% from normal values in 10 mildly affected animals(disease grade 1-2, see Experimental Procedures) and by 75±5% in 10severely affected rats (grade 4) compared to controls (FIG. 8B, 1 and2), attesting to the myasthenic nature of this animal model.Immunohistochemical staining with a polyclonal antiserum thatselectively detects AChE-R [Sternfeld et al. (2000) id ibid] revealedpositive signals in some, but not all muscle fibers of control rats.Similar patterns appeared under treatment with the inert, inverselyoriented oligonucleotide r-invEN102 (see FIG. 8B, 3). In EAMG rats (FIG.8B, 4), staining of AChE-R showed it as more generally distributed, withthe dispersed cytoplasmic localization that is characteristic of thisisoform [Soreq, H., and Seidman, S. (2001) Reviews Neuroscience 2,294-302], contrasting with the sub-synaptic cluster distribution of thesynaptic variant [Rossi, S. G. and Rotundo, R. L. (1993) J Biol Chem268, 19152-9]. Both the level of expression and the cellulardistribution of muscle AChE-S were similar in EAMG and healthy,untreated and r-invEN102-treated rats.

In situ hybridization using variant-selective probes showed that AChE-SmRNA was sub-synaptically located in muscles from both untreated andr-invEN102-treated, healthy and EAMG rats (FIG. 8B, 5 and 6). Incontrast, healthy rats displayed weaker and diffuse labeling of theAChE-R mRNA transcript, whereas a more pronounced punctuate labeling ofAChE-R mRNA appeared in triceps muscles of EAMG rats, unaffected byr-invEN102 treatment (FIG. 8B, 7 and 8). This accumulation in regionsrich in densely clustered nuclei was consistent with previousobservations of sub-synaptic regions [Rossi and Rotundo (1993) id ibid].These data indicate a selective over-expression of AChE-R in muscles ofEAMG rats and strengthened the idea of a role for this enzyme variant inMG pathophysiology.

Example 2 AChE-R and AChE-R mRNA Levels in Muscle Respond to rEN101

The soluble and secretory nature of AChE-R predicted that it woulddegrade acetylcholine before it reaches the post-synaptic membrane,limiting receptor activation. To test this hypothesis, rEN101 antisenseoligonucleotide was used, which is capable of selective suppression ofAChE-R production [Galyam, N. et al. (2001) Antisense Nucl Acid Drug Dev11, 51-57]. AChE-R suppression was tested in healthy and EAMG rats withreduced muscle nAChR levels (FIG. 8B, 1 and 2) 24 h after a single i.v.injection of 250 g/Kg rEN101. Immunohistochemical staining demonstratedthat AChE-R, but not AChE-S, was significantly reduced in muscles fromboth healthy and EAMG rats (FIG. 8C, 3 and 4 and data not shown).Receptor labeling patterns remained high in healthy rats and low in EAMGanimals, similar to those of untreated animals and animals treated withr-invEN102 (compare FIG. 8B, 1 and 2 to FIG. 8C, 1 and 2). In situhybridization indicated that AChE-S mRNA labeling, limited to the sitesof subsynaptic clusters of nuclei, was only nominally affected byrEN101, suggesting that neuromuscular transmission would be unaffectedby this treatment (FIG. 8C, 5 and 6). In contrast, rEN101 reduced AChE-RmRNA labeling almost to the limit of detection in both healthy andmyasthenic rats (FIG. 8C, 7 and 8).

Example 3 Suppression of AChE-R Restores Normal CMAP in EAMG Rats

Quantification by densitometry of an immunoblot analysis confirmed theincrease of serum AChE-R in EAMG and the efficacy of a single i.v.injection of 250 μg/Kg rEN101, but not r-invEN102, in reducing its serumlevel 24 h later (FIG. 9A). To evaluate the physiological outcome ofthis suppression, compound muscle action potentials (CMAPs) from thegastrocnemius muscle were recorded. EAMG rats, but never healthyanimals, displayed a decline in CMAP during repeated stimulation at 3Hz. The baseline decline, the percent difference in the heights of thefifth and the first evoked potentials, ranged from 10% to 36%(mean±SEM=13.0±2.5%, FIG. 9B, inset) as compared to 4.0±0.9% amonghealthy rats. The standard therapy for MG patients is administration ofanti-cholinesterases, which elevate ACh levels to a threshold thatenables receptor activation. Accordingly, neostigmine bromide(Prostigmine™, 75 μg/kg) was administered via i.p. This rapidly andeffectively corrected the CMAP decline in EAMG rats, from 87.6% of thefirst evoked potential in untreated animals to over 120% of this level(i.e. 107.4%) of the first evoked potential). The effects of thecholinesterase blockade were evident starting 15 min after the injectionand lasted 2 h, after which time the CMAP value returned to the baseline(FIG. 9B).

Unlike anticholinesterases, which block all AChE variants, rEN101 wasshown to selectively suppress muscle AChE-R production [Lev-Lehman etal. (2000) id ibid]. Therefore, retrieval of stable CMAP inrEN101-treated EAMG rats may attest to the causal role of AChE-R in theneuromuscular malfunctioning that is characteristic of the myasthenicphenotype. To test this concept, rEN101 was injected i.v. at dosesranging from 10-500 μg/Kg (2 to 20 nmol/rat). rEN101 did not affect CMAPin healthy animals, but retrieved stable CMAP ratios within 1 h (FIG.9B, inset, 9C and Table 1). CMAP normalization was accompanied byincreased mobility, upright posture, stronger grip, and reducedtremulousness of ambulation.

TABLE 1 Post-treatment CMAP ratios^(a) Oral^(b) Intravenous^(b)Phenotype EAMG EAMG Naive EAMG EAMG EAMG Treatment Naive EN101 EAMGEN101 EAMG EN102 invEN102 pyridostigmine EN101 EN101 EN102 invEN102 0 h1.01 ± 0.01 0.84 ± 0.03 0.82 ± 0.02 0.78 ± 0.06 0.90 ± 0.01 1.00 ± 0.0 0.87 ± 0.01 0.85 ± 0.06 0.89 ± 0.02 (4) (8) (4) (4) (6) (6) (6) (4) (5) 1 h^(c)  1.0 ± 0.02 0.97 ± 0.02 0.86 ± 0.04 0.86 ± 0.05 0.98 ± 0.010.02 ± 0.01 1.00 ± 0.01 1.04 ± 0.01 0.89 ± 0.03 (4) (8) (3) (4) (6) (7)(4) (4) (5) 5 h 1.03 ± 0.02 0.97 ± 0.03 0.96 ± 0.02 0.86 ± 0.05 0.96 ±0.02 1.00 ± 0.01 0.98 ± 0.02 0.98 ± 0.03 0.89 ± 0.02 (4) (7) (4) (4) (6)(6) (4) (4) (5) 24 h  1.01 ± 0.00 1.01 ± 0.01 0.95 ± 0.03 0.81 ± 0.080.87 ± 0.02 1.02 ± 0.01 1.00 ± 0.00 1.00 ± 0.01 0.90 ± 0.02 (7) (6) (5)(4) (6) (6) (4) (4) (5) ^(a)CMAP ratios (5^(th) vs. 1^(st) amplitude)were determined at the noted times following treatment. The averages ±SEM are presented. Each treatment represents similarly, although notsimultaneously treated rats, the numbers of which are shown inparentheses. ^(b)Drug doses were 50 μg/Kg for rEN101, rEN102 orr-invEN102 and 1000 μg/Kg for pyridostigmine (Mestinon Bromide), forboth administration routes. ^(c)Note the apparently delayed effect oforally administered rEN102 as compared to rEN101 or pyridostigmine.

Both the extent and the duration of CMAP correction were dose dependent.For example, 500 μg/Kg conferred 72 h rectification of CMAP up to 125%of baseline, while 50 μg/Kg was effective for only 24 h. rEN102, a 3′protected AS-ON targeting a sequence unique to rAChE-R mRNA (previouslyreferred to as AS1) [Grifman and Soreq (1997) id ibid], induced similarrectification of CMAP decline in EAMG rats, confirming the relevance ofAChE-R as a contributing element to this effect. Comparable amounts ofr-invEN102, did not improve muscle function, attesting to the sequencespecificity of the AS-ON treatment (Table 1). Dose response curvesrevealed that up to 5 h following an injection, rEN101 produced asaturable response with IC₅₀ of <10 μg/Kg. This effect appeared to besuperimposed on a longer lasting and less concentration-dependenteffect, which showed no saturation in the range studied (FIG. 9D). Thisphenomenon possibly reflected the altered muscle and/or neuromuscularjunction properties under the stable CMAP retrieval afforded by rEN101.

Example 4 Antisense Prevention of AChE-R Accumulation Promotes Staminain EAMG Rats

Placed on a treadmill at 25 m/min, healthy rats ran for 23.0±3.0 min,after which time they displayed visible signs of fatigue. Starting at 5h, and for at least 24 h following administration of 250 μg/Kg rEN101,EAMG rats demonstrated improved performance on the treadmill. Runningtime increased from 247±35, 179±21 and 32±6 sec to 488±58, 500±193 and212±59 sec for animals at disease grades 2, 3 and 4, respectively(average values for 6-9 animals per group.) Healthy animals, incontrast, were not significantly affected by rEN101 injection (FIG. 10).

Others have demonstrated efficacy of orally administered 2′-oxymethylprotected AS-ON agents [Monia, B. P. (1997) Ciba Found. Symp. 209,107-119]. Therefore, the inventors tested this mode in the EAMG model.Based on their own findings, the inventors selected the dose of 50 μg/Kgof rEN101, which was administered to EAMG rats once a day via oralgavage, and CMAP was measured 1, 5, and 24 h later. This dose was aseffective as 25 μg/Kg administered i.v. (Table 1 and FIGS. 9 and 13).Orally administered rEN102 was also active in reversing CMAP decline,but its effects appeared somewhat delayed compared to rEN101. Oralpyridostigmine (1000 μg/kg) restored CMAP for up to several hours, whiler-invEN102 had no significant effect (Table 1).

Example 5 Oral Administration of Human EN101 to EAMG Rats

Human EN 101 (hEN101) (0.25 μg/g, single dose) was administered orallyto rats with EAMG of medium severity (score 2.5-3.5), which implied thesymptoms defined as “moderate” hereunder. The results are summarized inTable 1. Time from treatment is noted above; together with the treadmillrunning time in sec. Animals were inspected at each time point forevaluation of muscle weakness. The clinical status of the rats wasgraded according to: (0)—Without definite weakness (treadmill runningtime, 23±3 min); Mild (1)—weight loss >3% during a week, >10 min.running time on treadmill; Moderate (2)—moderate weakness accompanied byweak grip or cry with fatigue, weight loss of 5-10%, 3-5 min. running ontreadmill; Moderate-severe (3)-moderate to severe weakness, hunched backposture at rest, head down and forelimb digit flexed, tremulousambulation, 10% body weight loss, 1-2 min. run on treadmill); Severe(4)—severe general weakness, no cry or grip, treadmill running time <1min, weight loss >10%; Death (5). Each line represents an individualrat. It is to be noted that the clinical score (in parentheses) wasreduced in all of the treated animals, which reflects time improvement,and that running time was significantly increased for over and 24 hr formost of the animals and for two of the tested animals also at 48 h.

TABLE 2 Treadmill Performance Time (Clinical score) before Animal(basal) 5 h 24 h 48 h Effect 1 110 sec 150 sec 360 sec ND ++ (3)   (1) 2  0 (3.5)  70 sec  30 sec ND +− (3) 3 210 sec 300 sec 345 sec ND ++(2.5) (1) 4 80 (3)  ND 170 sec  85 (2.5) +− (2) 5 180 (2.5)  |||| 380 290 (2)  ++ (1) 6  30 (3.5) 120 |||| (3) || (5) +These results show that like the rat EN101 (see treadmill example,above), the human EN101 antisense oligodeoxynucleotide of the inventionpromoted muscle stamina in EAMG induced rats.

Example 6 Comparative Analysis of hEN101 and rEN101 in a Rat AnimalModel

A study of the potential efficacy as well as toxicity of hEN101 wasconducted on 4 week-old Crl:CD rats. To groups of 12 animals (6 males, 6females) were administered 0.0 (saline only), 0.50 or 2.50 μg/g/day ofhEN101 by oral gavage (o.g.), 0.50 μg/g/day of rEN101 by o. g., or 0.50μg/g/day of rEN101 or hEN101 by i.v. injection. Additionally, there wasa control group that was not injected. For 7 days the animals werechecked for gross signs of toxicity: mortality, body weight, foodconsumption, ophtalmology, hematology (peripheral blood), and bloodchemistry. At 7 days they were sacrificed and examined post mortem formacroscopic pathology and organ weight. Fixed sections of brain(cerebellum, cerebrum, midbrain, medulla), heart (auricular andventricular regions), kidneys (cortex, medulla, papilla regions), liver(all main lobes), lungs (two major lobes, including bronchi), lymphnodes (mandibular and mesenteric), spinal cord (transverse andlongitudinal sections at cervical, lumbar and thoracic levels), caecum,colon, duodenum, ileum, jejunum, esophagus, rectum, spleen and stomach(keratinized, glandular and antrum) were stained with hematoxylin/eosinto reveal necrosis or cell death.

Mandibular lymph nodes were examined for the effect of EN101 on AChE-RmRNA. Compared to the saline-injected control, rEN101 (oral or i.v.) orhEN101 (i.v.) were inconsistent in depressing AChE-R mRNA levels withinthese lymph nodes (data not shown). In contrast, the administration ofrEN101 (oral or i.v.) or hEN101 (i.v.) did not affect the expression ofthe AChE-S synaptic variant of AChE in these mandibular lymph nodes(data not shown). Thus, the antisense oligodeoxynucleotide of theinvention may be used to suppress the AChE-R variant without affectingthe expression of the synaptic variant, i.e. without adversely affectingcholinergic transmission.

Example 7 AChE-R Suppression Modifies the Course of EAMG Pathophysiology

As shown in Example 5, unlike anti-cholinesterases, rEN101 affordedlong-term maintenance of stable CMAP. This further enabled the inventorsto test whether the cholinergic imbalance contributes to thephysiological deterioration that is characteristic of EAMG. Rats werefirst treated with rEN101 once a day for 5 days, CMAPs being determinedprior to each treatment. Both the efficacy of rEN101 in retrievingnormal CMAP and its capacity to reduce the inter-animal variability inCMAP values reached similar levels to those of pyridostigmine (FIGS. 11Aand 11B). However, the onset of response to pyridostigmine was morerapid (Table 1), while that observed with rEN101 was longer-lasting.Daily oral or i.v administration of rEN101 stabilized CMAPs over theentire course of treatment (FIG. 11B). In contrast, the effect ofpyridostigmine wore off within several hours, causing pronouncedfluctuations in muscle status (Table 1 and FIG. 11). Among the animalstreated daily with pyridostigmine, 5 out of 6 died within the 5 dayexperimental course. In contrast, 6 out of 8 animals treated once-a-daywith rEN101 via o.g. survived the full 5 day period. This conspicuousdifference might reflect the susceptibility of EAMG rats to repeatedanesthesia and CMAP measurements. In order to avoid these additionalstresses and evaluate the effect of the antisense treatment on EAMGpathophysiology, the inventors subjected groups of moderately sickanimals to 1 month of daily oral treatment with minimal interference.EAMG rats receiving oral doses of rEN101 daily, presented significantimprovement in survival, clinical status and treadmill performance, ascompared with pyridostigmine- and saline-treated animals (FIG. 12;P<0.041 for 4 weeks survival incidence, Fisher exact test, AS-ON vs.other treatments). One way repeated measures ANOVA yielded P<0.05 forall other measures (AS vs. other treatments at 4 weeks). The effect ofrEN101 on clinical symptoms was also corroborated by body weightchanges. Rats treated with saline and Mestinon treated groups lost 13.5and 11 g/animal, respectively, whereas animals treated with rEN101gained, on average, 13 g during the treatment period. Thus, daily rEN101administration promoted long-term change in the course of EAMG in ratswith moderate to severe symptoms, under the same conditions in whichuntreated or pyridostigmine-treated animals deteriorated.

By using MG and EAMG as case studies for evaluating the consequences ofchronic neuromuscular imbalance at the level of gene expression, theinventors confirmed that the AChE-R variant is systemically elevated inMG and EAMG. Moreover, the inventors showed that antisense suppressionof AChE-R normalized NMJ responses to repeated nerve stimulation,promoting muscle strength, and recuperating a healthier status inanimals otherwise too weak even to eat. These observations support theidea that AChE-R plays a direct role in MG pathophysiology and call forevaluation of the rationale of long-term mRNA-targeted therapy forimbalanced cholinergic function at NMJs.

Example 8 Oral Administration of hEN101 to Cynomolgus Monkeys

This experiment was conducted with six (3 males and 3 females)purpose-bred 15 month-old (young adult) Cynomolgus monkeys, divided inthree groups (1, 2 and 3) of one male and one female each. Groups 1 and2 received hEN101 daily by o.g. for a period of 7 days, at aconcentration of 0.15 and 0.50 μg/g/day, respectively. Group 3 receiveddaily i.v. injections of hEN101 for a period of 7 days at a dosage of0.50 μg/g/day.

Over a 12 hour period, plasma samples were obtained during the secondtreatment day to investigate the toxicokinetic profile at each dosage.The toxicology study consisted of checking the animals during the 7 daysof treatment for gross signs of toxicity by the following parameters:mortality, body weight, food consumption, electrocardiography, bloodpressure, hematology (peripheral blood), and blood chemistry. At 7 daysthe monkeys were sacrificed and examined post-mortem for macroscopicpathology and organ weight. Fixed sections of brain (cerebellum,cerebrum, midbrain, medulla), caecum, colon, duodenum, heart (auricularand ventriclar regions), ileum, jejunum, kidneys (cortex, medulla.papilla regions), liver (two main lobes), lungs (two major lobes,including bronchi), lymph nodes (mandibular and mesenteric), esophagus,rectum, sciatic nerve, skeletal muscle (thigh), spinal cord (transverseand longitudinal sections at cervical level), spleen and stomach (bodyand antrum) were stained with hematoxylin/eosin to reveal necrosis orcell death.

These clinical investigations revealed no toxicological effects by anyof the treatment protocols. Post-mortem there were no treatment-relatedeffects other than slight healing erosions in the body of the stomach,which are possibly associated with treatment, in 1 of 2 animals in eachgroup, and some irritation of the perivascular tissue at the site ofintravenous injection.

Paraffin-embedded 7 μm spinal cord sections were examined by in situhybridization for the levels of AChE-R and AChE-S mRNA. Under all threeregimens (oral 0.15 and 0.50, and i.v. 0.50 μg/g/day), there was noapparent reduction in AChE-S mRNA (FIG. 14). However, there was asignificant reduction in AChE-R mRNA in increasing hEN101 daily dosefrom 0.15 (oral) to 0.50 (oral or i.v.) with i.v. dosage being moreeffective.

AChE-R-positive sections from monkey spinal cords were analyzed for therelation between cell body diameter and percentage of AChE-R positivecells (FIG. 15). Cells were divided into three categories according totheir body diameter, and the percentage of AChE-R-positive cells fromeach category was evaluated. Treatment with the lower concentration ofEN101 (150 μg/kg/day) caused an increase in the percent of smallAChE-R-positive cells (<70 μm diameter) as compared to naive monkeys,probably due to the injection stress response, which is known to raiseAChE-R mRNA levels (Kaufer et al, 1998). Either i.v. or p.o.administration of the higher EN101 concentration (500 μg/kg/day) reducedthe percentage of AChE-R-positive neurons, compared to the lowerconcentration (p<0.05, Student's t test). The decrease was moreremarkable in small neurons (23-40 μm) than in neurons with cell bodydiameter of 40-70 μm, and no decrease was observed in large neurons (>70μm diameter) (FIG. 15). The percentage of small- (23 to 40 μm-diameter)and medium-sized (40 to 70 μm) neurons that were labeled decreasedsignificantly in moving from the lower to the higher hEN101 oral dose,and even further with the i.v. administration. Among the larger neurons(>70 μm), there was not discernable effect of hEN101. We have yet todiscover the functional correlate of cell size that determines theefficacy of antisense suppression of AChE-R expression. This suggeststhat EN101 will prevent the stress-induced impairment in interneuronsinput to motoneurons, thus preventing paralysis—e.g. post-surgery.

Example 9 hEN101 Suppression of AChE Activity in Monkey Plasma

In the 12 hr following the second day administration of hEN101, monkeyplasma samples were collected and stored. Plasma cholinesteraseactivities were measured by spectrophotometry assessing the rate ofhydrolysis of acetylthiocholine (measured by the Ellman assay, whichquantifies the hydrolysis of acetylthiocholine) [Ellman, G. L., et al.(1961), Biochem. Pharmacol. 7, 88-95], in the absence or presence ofiso-OMPA (a selective butyrylcholinesterase, BChE, inhibitor). Totalactivity, largely due to serum BChE, was generally unchanged (FIG. 16A).When measured in the presence of 1×10⁻⁵ M iso-OMPA, AChE activityincreased within the 5 hr post-injection. This increase, observed under150 μg/kg was effectively suppressed or attenuated by the higher dose of500 μg/kg hEN101, and even more effective when this dose was i.v.administered (FIG. 16B).

Example 10 Effect of rEN101 on Expression AChE-R mRNA in Rat Spinal CordNeurons

Contrary to the effect of hEN101 on monkey spinal cord neurons, rEN101does not suppress AChE-R mRNA in the rat spinal cord (FIG. 17), whenassessed by in situ hybridization using a mouse probe. Neither thenumber of positive cells nor the staining intensity were significantlychanged. One explanation for this result would be that the blood-brainbarrier that isolates the CNS is more permeable in monkeys than rats, atleast under the chosen experimental conditions.

Example 11 hEN101 Phase Ib Clinical Trial

16 patients with stable generalized MG requiring constant AChEinhibitors (pyridostigmine) for daily function were recruited, afterapproval by human ethics committees of the respective hospitals involvedin the present trial (Hadassah Medical Center, Jerusalem, Israel andGreater Manchester Neurosciences Center, Hope Hospital, Salford,England).

Analysis of the results obtained from the Clinical trial showed that in15 out of 16 patients, the initial deterioration in MG status afterstopping pyridostigmine was followed by a clear symptomatic improvementdue to hEN101. As an example, FIG. 18 shows the improvement in ptosis inone of the MG patients. Analysis of the mean daily QMG scores showed acontinuous decrease in each of the study days (decrease in QMG meansimprovement in disease status) (FIGS. 21A and 21B)., The baseline(entry) mean total score was 13.2. The score decreased for days 2through 6 in the amounts of 3.0, 4.8, 5.7, 5.5 and 6.0 (p<0.001). Themean percent improvement of total QMG for these days ranged from 27.8%to 53.4% (FIG. 20) (p<0.01). All individual test item scores, except forvital capacity and left arm out stretched time, had statisticallysignificant change from entry for days 2-6 (p<0.05). Improved QCG scoresfollowing the final dose of hEN101 were sustained for up to 72 hours. Noserious adverse effects were observed. Vital signs, chemical chemistry,hematology, urinalysis, ECG and physical examination remained unchangedthroughout the experimental period and in the month following discharge.Cholinergic side effects were not reported.

Thus, hEN101 appears to be powerfully effective in reversing symptoms inpatients with stable MG. The present study showed that hEN101 haspotential advantages over conventional cholinesterase inhibitors withrespect to dosing, specificity, side-effect profile, duration ofefficacy and treatment regimen.

1. A synthetic antisense oligodeoxynucleotide targeted against humanAChE mRNA having the nucleotide sequence: 5′ CTGCCACGTTCTCCTGCACC 3′.(SEQ ID NO: 1)


2. A synthetic nuclease resistant antisense oligodeoxynucleotide havingthe nucleotide sequence: 5′ CTGCCACGTTCTCCTGCACC 3′. (SEQ ID NO: 1)


3. A synthetic antisense oligodeoxynucleotide of claim 2, which is amodified oligodeoxynucleotide comprising partially unsaturated aliphatichydrocarbon chain and one or more polar or charged groups includingcarboxylic acid groups, ester groups, and alcohol groups.
 4. A syntheticnuclease resistant antisense oligodeoxynucleotide of claim 2 or 3,wherein at least one of the three 3′-terminus nucleotides is2′-O-methylated.
 5. A synthetic nuclease resistant antisenseoligodeoxynucleotide of claim 4, in which the last three 3′-terminusnucleotides are 2′-O-methylated.
 6. A synthetic nuclease resistantantisense oligodeoxynucleotide of claim 2, wherein at least onenucleotide is fluoridated.
 7. A synthetic nuclease resistant antisenseoligodeoxynucleotide of claim 2 or claim 3, having phosphorothioatebonds linking between at least two of the last 3′-terminus nucleotidebases.
 8. A synthetic nuclease resistant antisense oligodeoxynucleotideof claim 7, having phosphorothioate bonds linking between the last four3′-terminus nucleotide bases.
 9. A synthetic nuclease resistantantisense oligodeoxynucleotide of claim 3, having a nucleotide loopforming sequence at the 3′-terminus.
 10. A synthetic nuclease resistantantisense oligodeoxynucleotide of claim 9, wherein said loop is a9-nucleotide loop having the nucleotide sequence CGCGAAGCG (SEQ IDNO:2).
 11. A synthetic nuclease resistant antisense oligodeoxynucleotideof any one of claims 1 to 10, capable of selectively modulating humanAChE production.
 12. A synthetic nuclease resistant antisenseoligodeoxynucleotide of claim 11, capable of selectively modulatinghuman AChE production in the central nervous system.
 13. Apharmaceutical composition comprising the antisense oligonucleotidehEN101, defined by SEQ ID NO:1.
 14. The pharmaceutical composition ofclaim 13, for the treatment and/or prevention of a progressiveneuromuscular disorder, for improving stamina and/or for use in chronicmuscle fatigue.
 15. The pharmaceutical composition of claim 13 for usein treating or preventing a progressive neuromuscular disorder, whereinsaid disorder is selected from myasthenia gravis, Eaton-Lambert disease,muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stressdisorder (PTSD), multiple sclerosis, dystonia, post-stroke sclerosis,post-injury muscle damage, excessive re-innervation, post-surgeryparalysis of unknown origin, and post-exposure to AChE inhibitors. 16.The pharmaceutical composition of claim 13, for the treatment and/orprevention of myasthenia gravis.
 17. The pharmaceutical composition ofclaim 13, for use in treating or preventing a progressive neuromusculardisorder, wherein said disorder is associated with an excess of AChEmRNA or protein.
 18. The pharmaceutical composition of claim 13, for usein treating or preventing a progressive neuromuscular disorder, whereinsaid disorder is associated with an excess of AChE-R mRNA.
 19. Thepharmaceutical composition of claim 13, for use in treating orpreventing a progressive neuromuscular disorder, wherein said disorderis associated with impairment of cholinergic transmission.
 20. Thepharmaceutical composition of claim 13, for use in treating orpreventing a progressive neuromuscular disorder, wherein said disorderinvolves muscle distortion, muscle re-innervation or neuro-muscularjunction (NMJ) abnormalities.
 21. The pharmaceutical composition of anyone of claims 13-20, which is for daily use by a patient of a dosage ofactive ingredient between about 0.001 μg/g and about 50 μg/g.
 22. Thepharmaceutical composition of anyone of claims 13-20, wherein thetreatment and/or prevention comprises administering a dosage of activeingredient of about 0.01 to about 5.0 μg/g.
 23. The pharmaceuticalcomposition of any one of claims 13-20, wherein the treatment and/orprevention comprises administering a dosage of active ingredient ofabout 0.15 to about 0.50% g/g.
 24. The pharmaceutical composition ofclaim 13, optionally comprising at least one additional active agent.25. A pharmaceutical composition comprising an antisenseoligodeoxynucleotide as denoted by SEQ ID NO:1, for facilitating passageof compounds through the BBB, optionally further comprising additionalpharmaceutically active agent to be transported through the BBB, and/orpharmaceutically acceptable adjuvant, carrier or diluent.
 26. Thepharmaceutical composition of claim 25, wherein said additionalpharmaceutically active agent is selected from any one of contrastagents used for central nervous system imaging, agents that function toblock the effects of abused drugs, antibiotics, chemotherapeutic drugsand vectors to be used in gene therapy.
 27. A method of preparation of apharmaceutical composition comprising the step of admixing the antisenseoligonucleotide hEN101, defined by SEQ ID NO:1, with a pharmaceuticallyacceptable adjuvant, carrier or diluent, and optionally with at leastone additional active agent.
 28. The method of claim 27, wherein saidcomposition is intended for the treatment and/or prevention of aprogressive neuromuscular disorder, for improving stamina and/or for usein chronic muscle fatigue.